Most bacterial viruses (bacteriophages or just phages) are highly efficient in their ability to infect their often very specific bacterial hosts In general, only one or a few viral particles are necessary to successfully infect one bacterium. As a consequence there has been a long, unfulfilled hope that phages could be used as antibiotics. The need for alternative antibiotics is becoming ever more acute as common bacterial pathogens are developing resistant mutations, making it urgent to develop alternatives such as phage therapy. In addition, recombinant phages have been suggested as a means for gene therapy and as potential antigens for development of vaccines. Bacteriophage T4 has been a model system to study virus structure and function, having advanced biochemical principles of the assembly of biological complexes and protein-protein interactions. We have extensive experience in both structural and functional studies of phage T4. We now wish to expand this knowledge and make it available for potential medical applications. The plan is to analyze the structure and assembly of the capsid (Specific Aim 1), recognition of the host by the phage fibers (Specific Aim 2), the DNA packaging machine (Specific Aim 3), attachment of the packaging machine to the head (Specific Aim 4), and the mechanism of gene delivery (Specific Aim 5). These studies are directed towards engineering phage T4 for specific medical applications to target specific prokaryotic cells (as an antibiotic) and eukaryotic cells (such as cancer cells) for the delivery of toxins. In addition, we are engineering the surface of T4 to carr multiple copies of an epitope for vaccine development. Our primary tools will be molecular biology, protein chemistry, crystallography and electron microscopy. Molecular biology studies will produce pure samples in sufficient quantity for structural and functional studies. Crystallographic studies will be of protein components, providing three-dimensional information at near atomic resolution. Cryo-electron microscopic reconstructions will provide three-dimensional data on the organization of the protein components within the virus. Combining these techniques will generate pseudo-atomic resolution structures of the virus at different stages of its life cycle for functional interpretation and verification.